Hypotransferrinemic (Trf(hpx/hpx)) mice have a severe deficiency in serum transferrin (Trf) as the result of a spontaneous mutation linked to the murine Trf locus. They are born alive, but before weaning, die from severe anemia if they are not treated with exogenous Trf or red blood cell transfusions. We have determined the molecular basis of the hpx mutation. It results from a single point mutation, which alters an invariable nucleotide in the splice donor site after exon 16 of the Trf gene. No normal Trf messenger RNA (mRNA) is made from the hpx allele. A small amount of mRNA results from the usage of cryptic splice sites within exon 16. The predominant cryptic splice site produces a Trf mRNA carrying a 27-base pair (bp), in-frame deletion. Less than 1% of normal levels of a Trf-like protein is found in the serum of Trf(hpx/hpx) mice, most likely resulting from translation of the internally deleted mRNA. Despite their severe Trf deficiency, however, Trf(hpx/hpx) mice initially treated with transferrin injections can survive after weaning without any further treatment. They have massive tissue iron overload develop in all nonhematopoietic tissues, while they continue to have severe iron deficiency anemia. Their liver iron burden is 100-fold greater than that of wild-type mice and 15- to 20-fold more than that of mice lacking the hemochromatosis gene, Hfe. Trf(hpx/hpx) mice thus provide an additional model with a defined molecular defect for the study of genetic iron disorders.